Mechanisms of endometrial progesterone resistance

Molecular and Cellular Endocrinology 358 (2012) 208–215

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Molecular and Cellular Endocrinology journal homepage: www.elsevier.com/locate/mce

Review

Mechanisms of endometrial progesterone resistance Marwa Al-Sabbagh b, Eric W.-F. Lam a, Jan J. Brosens c,⇑ a

Cancer Research-UK Labs and Department of Surgery and Cancer, Imperial College London, Hammersmith Hospital, London, United Kingdom Institute of Reproductive and Developmental Biology, Department of Surgery and Cancer, Imperial College London, Hammersmith Hospital, London, United Kingdom c Division of Reproductive Health, Warwick Medical School, Clinical Sciences Research Laboratories, University Hospital, Coventry CV2 2DX, United Kingdom b

a r t i c l e

i n f o

Article history: Available online 9 November 2011 Keywords: Endometriosis Endometrium Progesterone receptor Coregulator Transcription Posttranslational modification Chromatin Epigenetics Menstruation

a b s t r a c t Throughout the reproductive years, the rise and fall in ovarian hormones elicit in the endometrium waves of cell proliferation, differentiation, recruitment of inflammatory cells, apoptosis, tissue breakdown and regeneration. The activated progesterone receptor, a member of the superfamily of ligand-dependent transcription factors, is the master regulator of this intense tissue remodelling process in the uterus. Its activity is tightly regulated by interaction with cell-specific transcription factors and coregulators as well as by specific posttranslational modifications that respond dynamically to a variety of environmental and inflammatory signals. Endometriosis, a chronic inflammatory disorder, disrupts coordinated progesterone responses throughout the reproductive tract, including in the endometrium. This phenomenon is increasingly referred to as ‘progesterone resistance’. Emerging evidence suggests that progesterone resistance in endometriosis is not just a consequence of perturbed progesterone signal transduction caused by chronic inflammation but associated with epigenetic chromatin changes that determine the intrinsic responsiveness of endometrial cells to differentiation cues. Ó 2011 Elsevier Ireland Ltd. All rights reserved.

Contents 1. 2. 3. 4. 5.

6. 7.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Progesterone actions in the human endometrium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Progesterone resistance in the endometrium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Progesterone receptor signalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell- and environment-specific progesterone receptor signalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Progesterone receptor coregulators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Crosstalk between the progesterone receptor and other co-transcription factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Posttranslational modifications of the progesterone receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. Dynamic interactions with the chromatin landscape . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mechanisms of endometrial progesterone resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of Interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Endometriosis is clinically defined by the presence of visible endometrial implants outside the uterus. It is widely viewed as an oestrogen-dependent disorder (Bulun, 2009), which seems log-

⇑ Corresponding author. Tel.: +44 24 7696 8704. E-mail address: [email protected] (J.J. Brosens). 0303-7207/$ - see front matter Ó 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2011.10.035

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ical as endometrial tissue, ectopic or not, involutes in the absence of oestrogens. Whether or not aberrant local oestrogens production and signalling are genuinely causal to endometriosis remains unresolved. After all, medical interventions based on oestrogens suppression may be useful in alleviating pain and other symptoms associated with endometriosis but do not cure the disease. It is important to emphasize that the clinical definition of endometriosis is narrow and perhaps even misleading. As a disease, endometriosis extends well beyond the presence of

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ectopic implants and impacts on the different hormone responsive compartments in the female reproductive tract, including the ovary, fallopian tube, the uterine junctional zone and, of course, the endometrium (Brosens et al., 2010; Giudice and Kao, 2004; Gupta et al., 2008; Kunz et al., 2005). Of all these structures, the endometrium is by far the most accessible. In recent years, a body of molecular evidence has emerged indicating that the endometrial responses to the postovulatory surge in circulating progesterone differ profoundly between patients with endometriosis and healthy controls. Hence, the phrase ‘progesterone resistance’ was coined to describe the deregulation of differentiation-specific gene networks in the endometrium in patients with endometriosis (Aghajanova et al., 2010; Bulun, 2009; Bulun et al., 2006; Fazleabas, 2010). At first glance, the terms ‘oestrogens-dependence’ and ‘progesterone-resistance’ appear to describe opposite sides of the same coin. It is however important to keep in mind that all key reproductive events controlled by progesterone, including ovulation, embryo implantation, decidualization, and menstrual shedding, require influx of distinct immune cells and controlled local inflammation (Brosens and Gellersen, 2006; Brosens et al., 2009; Gellersen et al., 2007). In other words, the physiological responses to progesterone, unlike those to estradiol, are strictly intertwined with activation of inflammatory pathways. A salient feature of endometriosis is chronic pelvic inflammation and oxidative stress, which in turn represent powerful cues capable of modulating progesterone responses in target tissues. This review summarizes our current understanding of progesterone actions in the endometrium. In addition, we examine the mechanisms that govern the cellular responses to progesterone and other differentiation cues and explore how perturbations in these systems may contribute to the pathogenesis of endometriosis as a chronic inflammatory disease.

2. Progesterone actions in the human endometrium Progesterone elicits a broad spectrum of responses in the female reproductive tract as well as in extra-reproductive tissues, such as bone, cardiovascular and respiratory systems, kidney, adipose tissue, and the brain (Gellersen et al., 2009; Graham and Clarke, 1997). The ovary is not only the major source of progesterone production during the cycle but progesterone signalling is implicated in follicular growth, ovulation and luteinization. Progesterone also has profound effects on the contraction waves of the junctional zone, on tubal transport function, and on cervical secretion. However, the most studied target tissue is the endometrium. Here, the postovulatory surge in progesterone triggers a highly coordinated and sequential responses, commencing with arrest of oestrogens-dependent epithelial cell proliferation and followed by the secretory transformation of the glands, recruitment of various bone marrow-derived immune cells, and angiogenesis (Brosens et al., 1999; Gellersen et al., 2007; Gellersen and Brosens, 2003). At a functional level, progesterone transiently induces a receptive phenotype in luminal endometrial epithelial cells, essential for embryo implantation (Brosens et al., 2009a; Dey et al., 2004), although this response is mediated by signals derived from the underlying differentiating stromal cells (Simon et al., 2009). This differentiation process in the stromal compartment is termed ‘decidualization’ and characterized by the transformation of endometrial stromal cells into specialized secretory decidual cells (Cloke et al., 2008; Dey et al., 2004; Gellersen et al., 2007). In most mammals with an invasive placenta, decidualization of the endometrial stromal compartment is initiated at the site of implantation in response to embryonic signals. Spontaneous decidualization of the stromal compartment in the absence of pregnancy is a rare biological phenomenon, which, like spontaneous

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endometriosis, is confined to humans and the few other menstruating species (Brosens et al., 2009b, 2002; Jabbour et al., 2006). Once the endometrium undergoes a decidual response, the integrity of the tissue becomes inextricably dependent upon continuous progesterone signalling. In the absence of pregnancy, declining progesterone levels triggers a switch in the secretory repertoire of decidual stromal cells, now characterized by expression of pro-inflammatory cytokines, chemokines and matrix metalloproteinases, which activates a sequence of events leading to tissue breakdown of the superficial endometrial layer, focal bleeding and menstrual shedding (Brosens and Gellersen, 2006; Brosens et al., 2009; Brun et al., 2009; Gaide Chevronnay et al., 2009; Kokorine et al., 1996; Marbaix et al., 1995). Decidualization of endometrial stromal cells is an intriguing example of mesemchymal-epithelial transition (MET), a reversible biological process that bestows an epitheloid phenotype on spindle-shaped mesenchymal cells. Consequently, decidualizing stromal cells express a myriad of genes that are otherwise only expressed in the epithelial cell compartment of the endometrium (Takano et al., 2007). Although MET is much less studied than the reverse process, epithelial-mesenchymal transition (EMT), it is believed to participate in stabilization of distant metastases by allowing cancerous cells to regain epithelial properties and integrate into distant organs (Yang and Weinberg, 2008). MET is also an obligatory event in pluripotent stem cell programming of mouse fibroblast upon ectopic expression of Oct4, Klf4, Sox2, and c-Myc (Li et al., 2010; Samavarchi-Tehrani et al., 2010). Interestingly, mining of microarray data revealed that differentiation of endometrial fibroblast into decidualizing cells is associated with enhanced expression of three of these four genes (OCT4, KLF4, and MYC) (Takano et al., 2007). The decidual phenotype is intriguing not only for the apparent cellular plasticity. Decidualization seems to bestow some seemingly conflicting properties on endometrial cells, such as the ability to undergo apoptosis upon progesterone withdrawal but also to resist oxidative stress cell death, to regulate local immune responses, and to enhance the invasiveness of stromal cells (Gellersen et al., 2010; Labied et al., 2006; Leitao et al., 2010, 2011). Intriguingly, these specific functions of decidual cells are also attributes implicated in the formation of ectopic implants upon retrograde menstruation.

3. Progesterone resistance in the endometrium As mentioned, the term ‘progesterone resistance’ derived from the observation that a subset of progesterone-dependent genes in the eutopic secretory endometrium is deregulated in endometriosis patients (Aghajanova et al., 2010; Burney et al., 2007; Kao et al., 2003; Taylor et al., 1999). Progesterone resistance is also a hallmark of ectopic implants and probably plays a role in ovarian and tubal dysfunction associated with the endometriosis (Bulun, 2009; Wu et al., 2006a,b). Initial microarray profiling study identified in excess of 200 dysregulated genes in mid-secretory biopsies from women with minimal or mild endometriosis compared to disease-free individuals (Kao et al., 2003). A subsequent study showed that impaired gene expression in eutopic endometrium of patients with endometriosis occurs throughout the entire cycle, including the proliferative phase, although the most extensive changes were found in early-secretory endometrium (Burney et al., 2007). Many of the deregulated genes identified during this phase of the cycle were known progesterone targets and the overall pattern of aberrant gene expression suggested a prolongation of the proliferative phenotype after ovulation (Burney et al., 2007). At first glance, the concept of uterine progesterone resistance in endometriosis is not only easy to understand, it also seems to

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provide a very plausible biological explanation for a link between pelvic endometriosis and conception delay or infertility. On closer scrutiny, however, there are several problems with the concept. First, studies on purified eutopic endometrial stromal cells from patients with endometriosis demonstrated that the aberrant pattern of gene expression is not confined to progesterone stimulation. In a recent microarray study, no fewer than 245 genes were reportedly differentially expressed in unstimulated primary endometrial stromal cell cultures established from endometriosis patients and controls (Aghajanova et al., 2010). In addition to basal gene expression and progesterone responses, endometriosis is further associated with perturbed gene expression upon stimulation with cyclic adenosine monophosphate (cAMP) or human chorionic gonadotropin stimulation (Aghajanova et al., 2010; Sherwin et al., 2010). Thus, the term ‘progesterone resistance’ appears a misnomer, as multiple key endometrial signal transduction pathways seem affected in endometriosis. Second, abnormal behaviour of purified endometrial cells in culture is not specific for endometriosis. For example, the ability of endometrial stromal cells to express prolactin, a major marker of decidualization, is grossly abnormal in patients suffering from recurrent pregnancy loss (Salker et al., 2010). Purified eutopic endometrial cells from patients with uterine adenomyosis also display abnormal behaviour in culture, such as increased invasiveness (Mehasseb et al., 2010). Further, based on microarray analysis of timed endometrial biopsies from patients treated with either clomiphene citrate or progesterone supplements, the endometrium in polycystic ovary syndrome patients has also been labelled as being progesterone-resistant (Savaris et al., 2011). Finally, there is a clear mismatch between the level of progesterone resistance described in purified cultures and the clinical presentation of patients with endometriosis. Aghajanova and colleagues (Aghajanova et al., 2011) reported that progesterone treatment of purified endometrial stromal cells from disease-free patients alters the expression of 8, 62, and 172 genes after 6 h, 2 and 14 days of stimulation, respectively. Uterine stromal cells from patients with mild endometriosis, however, responded to the same treatment regime by inducing the expression of only 0, 3 and 4 genes, respectively (Aghajanova et al., 2011). When extrapolated to the in vivo situation, these observations suggest that the stromal compartment of the endometrium is completely refractory to progesterone signalling in patients with endometriosis, which in turn implies that pregnancy, if at all possible, would invariably result in miscarriage. In fact, there is no compelling clinical evidence to suggest that it increases the risk of miscarriage (Jacobson et al., 2010; Marcoux et al., 1997; Metzger et al., 1986). Thus, the concept of ‘progesterone resistance’, and its clinical relevance, is far from established and in need of refining. Furthermore, to be of translational relevance, this apparent cellular refractoriness to hormonal signalling must be defined at molecular level. In fact, not one but several mechanisms have been described, all of which converge on the nuclear progesterone receptors as described below.

4. Progesterone receptor signalling The progesterone responses are mediated primarily through binding to and activation of the nuclear receptors, PR-A and PRB, members of the superfamily of ligand-activated transcription factors (Misrahi et al., 1987). Upon progesterone binding, PR has the ability to directly bind to DNA and regulate the expression of target genes. However, progesterone can sometimes activate a variety of rapid signalling events, independently of transcriptional regulation or even in the absence of its nuclear receptors (Gellersen et al., 2009). It is widely accepted that these rapid non-genomic

signalling events together with the comparatively slower genomic actions that determine the functional response to progesterone in a cell type- and environment-specific manner. While the genomic actions are relatively well defined, in-depth mechanistic insights into non-genomic progesterone actions are largely lacking (Gellersen et al., 2009). As is the case for all other nuclear receptors, PR has a modular structure made up of distinct functional domains, which can be swapped between conserved receptors without a loss of function (Brosens et al., 2004; Kastner et al., 1990). Both PR isoforms derive from different promoter usage in a single gene but PR-B differs from PR-A in that it contains an additional 164 amino acids at the amino-terminus (Kastner et al., 1990). A number of alternatively transcribed, translated or spliced isoforms have been described, including PR-C, PR-M, or PR-S, however, it is unclear if these truncated PR variants are actually expressed at physiologically relevant levels in vivo (Samalecos and Gellersen, 2008). While PR-A and -B display indistinguishable hormone- and DNA-binding affinities, their actions are remarkably divergent. Early functional studies, using reporter assays driven by simple or complex progesterone response elements (PREs), indicated that the liganded PR-A has very limited intrinsic transcriptional activity, suggesting that it functions primarily as a dominant inhibitor of PR-B and various other steroid receptors, including the oestrogens receptor (Vegeto et al., 1993). However, this view is no longer tenable. For example, PR-A and -B were subsequently shown to govern distinct endogenous gene networks in progesterone-responsive cells (Richer et al., 2002). More importantly, selective gene ablation studies in mice revealed that only PR-A is indispensable for ovarian and uterine functions whereas PR-B but not -A is critical for mammary gland development (Conneely et al., 2002; Mulac-Jericevic et al., 2003). Like other steroid receptors, PR contains defined sequences, termed nuclear import and export signals, which enables the receptor to shuttle actively between the nuclear and cytoplasmic compartments. The unliganded receptor is assembled in a large multi subunit complex that contains various heat shock proteins (e.g. HSP90, HSP40, HSP70 and p23) and immunophilins (e.g. FKBP51 and FKBP52) (Kosano et al., 1998; Tranguch et al., 2007). These chaperone proteins maintain the receptor in conformation state that allows hormone binding and play a critical role in the dynamic shuttling of the receptor. Progesterone, which is lipophilic, can freely translocate through the cell membrane and trigger a conformational change in PR. This in turn results in dissociation from the chaperone proteins, dimerization and binding of the receptor to specific DNA recognition sequences in the promoters of target genes, leading to activation or repression of transcription (Brosens et al., 2004).

5. Cell- and environment-specific progesterone receptor signalling The classical model outlined above predicts that progesterone treatment of PR expressing cells will modulate, in concert, the expression of numerous genes with accessible DNA response elements in their promoter region. The model also implies that the level of the response is proportional to the abundance of the receptor in the cell. However, both predictions are not supported by experimental data. In reality the mechanism of progesterone receptor action is more complicated. For example, there are few, if any, genes regulated promptly by progesterone in purified primary endometrial cells, despite the presence of abundant receptors in these cells (Aghajanova et al., 2011). An example of this is decidualization, which denotes the differentiation of endometrial stromal cells into specialized decidual cells indispensable for pregnancy. While decidualization is unequivocally a progesterone-dependent

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process, endometrial stromal cells become sensitive to progesterone signalling only when the protein kinase A (PKA) pathway is first activated in response to rising intracellular cAMP levels (Brosens et al., 1999; Gellersen and Brosens, 2003; Jones et al., 2006). Endometrial cAMP levels also rise in vivo during the secretory phase of the cycle, which probably reflect the actions of a variety of factors, including prostaglandin E2, corticotropin releasing factor, and relaxin. In other words, additional signals are required to fine-tune progesterone activity to ensure that progesterone triggers an appropriate response in a particular cell within a specific environment. 5.1. Progesterone receptor coregulators Recruitment of the liganded PR to DNA is in itself insufficient to alter target gene expression. The reason for this is that nuclear receptors themselves do not possess the necessary chromatin remodelling enzymatic activity to modify the accessibility of the basal transcriptional machinery to the chromatin DNA (Han et al., 2009; Lonard and O’Malley, 2006; Thakur and Paramanik, 2009). To modulate gene expression, PR and other co-transcription factors must therefore first recruit coregulators with intrinsic histone- and DNA-modifying activities. The number of coregulators identified has grown exponentially since the discovery of the first such coregulator, termed steroid receptor coactivator-1 (SRC-1) and totals now over 300 different proteins (Onate et al., 1995; Thakur and Paramanik, 2009). Coregulators are broadly divided in coactivators and corepressors, depending on whether they promote or repress transcription, respectively; although for some this distinction is rather vague (Han et al., 2009; Lonard and O’Malley, 2006; Thakur and Paramanik, 2009). Based on their mechanisms of action, nuclear receptor coactivators can further be categorized into three major function complexes: (i) the SWI/SNF complex, which remodel the local chromatin structure through adenosine triphosphate-dependent histone acetylation; (ii) the SRC complex, which contain acetyltransferases (e.g. CBP, p300, and the p300/CBP-associated factor) and methyltransferases (e.g. CARM1 and PRMT1); and (iii) mediator complex involved in the activation of RNA polymerase II and transcriptional initiation. In addition to chromatin modification and remodelling, coactivators have been implicated in a variety of other processes, including initiation of transcription, elongation of RNA chains, mRNA splicing, and even, proteolyic termination of the transcriptional response (Thakur and Paramanik, 2009). The prototypic nuclear receptor corepressors are NCoR (nuclear receptor corepressor) and SMRT (silencing mediator for retinoid and thyroid hormone), which exist in large protein complexes that include histone deacetylases (Li et al., 2000; Li and O’Malley, 2003). Another putative corepressor, RIP140, promotes the assembly of DNA- and histone-methyltransferases upon interaction with DNA-bound nuclear receptors, which further indicates that transcriptional repression is mediated primarily through epigenetic mechanisms, including nucleosomal condensation and DNA methylation (Kiskinis et al., 2007). However, to date NCoR and SMRT have only been shown to interact with antagonist-bound PR. Whether they have a role in progesterone-dependent repression of target genes requires further clarification. 5.2. Crosstalk between the progesterone receptor and other cotranscription factors PR can regulate gene expression in concert with other co-transcription factors. This mechanism is referred to as transcription factor ‘crosstalk’ and is dependent on protein–protein interactions between steroid receptors and other sequence-specific transcription factors. Transcriptional crosstalk is critical for cycle-depen-

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dent endometrial differentiation. Initiation of the decidual response by cAMP involves activation of several transcription factors, including p53, FOXO1, HOXA10, HOXA11, STAT5, and C/EBPb, capable of interacting directly or indirectly with PR (Christian et al., 2011, 2002a,b,c; Lynch et al., 2009; Mak et al., 2002; Pohnke et al., 2004; Schneider-Merck et al., 2006). This has led to the concept that PR, and especially the A isoform which is indispensible for decidualization (Conneely et al., 2002), may function as a scaffold for the assembly of multimeric transcriptional complexes to regulate the expression of decidua-specific genes in endometrial cells. Like other steroid hormone receptors, PR can also modulate gene expression without interacting directly with DNA. Well-characterised examples include PR modulation of the Activator Protein 1 (AP1), Nuclear Factor-KappaB (NF-jB) and Specificity Protein 1 (SP1) transcription factors (Bamberger et al., 1996; Kalkhoven et al., 1996; Owen et al., 1998). In this way, by recruiting other transcription factors, the hormone-bound PR can exert its influence on a variety of genes that do not have consensus PR-binding elements in their promoters, thus expanding its transcriptional network. Intriguingly, many of these transcription factors are able to interact with the unliganded PR, accounting for the activity of the receptor in the absence of progesterone (Cloke et al., 2008). 5.3. Posttranslational modifications of the progesterone receptor The activity of PR, as well as that of its transcriptional partners and coregulators, is further modulated by a number of posttranslational modifications, including phosphorylation, sumoylation, ubiquitination, and acetylation (Abdel-Hafiz et al., 2002; Brosens et al., 1999; Daniel et al., 2010; Jones et al., 2006; Lange et al., 2000; Leitao et al., 2010). These modifications provide a rapid and dynamic mechanism to fine-tune the function as well as the activity of PR complex in response to changes in hormonal, growth factor, cytokine, and environmental stress signals. For example, PR has numerous phosphorylation sites, at least 14 of which have been characterised. Some of these sites are modified in response to hormone binding and others by kinases, including mitogen-activated protein kinase (MAPK), casein kinase II, and cyclin-dependent protein kinase-2, upon growth factor signalling (Dressing et al., 2009). The phosphorylation state of PR influences its subcellular localization, transcriptional activity, protein stability, interaction with other cofactors, and target-gene specificity. An important feature of the activated PR in both endometrial and breast cells is that it regulates the expression of several genes that encode for intermediates of various signal transduction pathways, including the WNT/b-catenin, TGFb/SMAD and STAT pathways (Cloke et al., 2008; Li and O’Malley, 2003). Thus, progesterone is capable of reprogramming the activity of other signal transduction pathways, which in turn can alter its own posttranslational modification code and that of its coregulators and co-transcription factors. Sumoylation has emerged as another critical modification system that determines the activity of PR in the reproductive tract (Abdel-Hafiz et al., 2009; Abdel-Hafiz et al., 2002; Jones et al., 2006; Leitao et al., 2010; Leitao et al., 2011). Like ubiquitination, sumoylation denotes a process in which target proteins, mostly transcription factors, are modified by covalent attachment of a small peptide, SUMO (small ubiquitin modifier), in an enzymatic reaction. While ubiquitination frequently earmarks proteins for proteasomal degradation and intracellular transport, binding of SUMO generally bestows transcription factors with a wild range of properties. For instance, mutation of the single SUMO binding site in PR-A into a sumoylation-deficient receptor converts this PR isoform from a weak to a potent transcriptional activator. Interestingly, cAMP signalling in endometrial stromal cells alters the expression of many conjugating and de-conjugating SUMO enzymes, resulting in a reduction in PR-A sumoylation and increased

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receptor activity (Jones et al., 2006). The SUMO pathway is also extremely sensitive to a variety of environmental stress signals, including oxidative stress. In endometrial cells, relative low levels of oxygen free radicals are sufficient to promote global as well as PR-specific sumoylation, leading to loss of receptor activity. Strikingly, this coupling of oxidative stress signals to PR via enhanced sumoylation is disabled upon differentiation of endometrial stromal cells into decidual cells (Leitao et al., 2010, 2011). 5.4. Dynamic interactions with the chromatin landscape Gene regulation by steroid hormones like progesterone is a comparatively slower process that takes several hours and sometimes days. At first glance, the slow kinetics of the response agrees well with the concept that the activated PR must first bind to the promoter of target genes, recruit coregulators, assemble them in a multimeric complex that has the right enzymatic activity to modify the local chromatin structure, which in turn will lead to changes in the transcriptional machinery, efficacy of RNA synthesis, translation and, ultimately, protein levels. This static model is being profoundly challenged by novel techniques that allow genome-wide mapping of binding of nuclear receptors to DNA and real-time

monitoring of transcription (Biddie et al., 2010; George et al., 2009; John et al., 2011). First the interaction of nuclear receptors and other transcription factors with the chromatin turned out to be a highly dynamic process, characterized by rapid cycles, measured in seconds of transient association and dissociation with the chromatin (McNally et al., 2000), which in turn leads to oscillating transcriptional events. Receptor turnover by ubiquitination and chaperone proteins are indispensable for this cyclic recruitment of PR to the chromatin template. Moreover, the nature of the ligand determines the kinetics of interaction with chromatin, characterized by rapid exchanges with agonist-bound PR but much slower interactions upon treatment with antagonists like RU486 (Rayasam et al., 2005). Second, genome-wide mapping has revealed that the majority of nuclear receptors bind response elements located at a considerable distances from target promoters. Moreover, rather than the activated receptor inducing local chromatin remodelling to allow transcription, many of these sites are already ‘pre-existing’ or constitutively accessible (Biddie et al., 2010; John et al., 2011). These observations indicate not only that long-range interactions between distant regulatory elements and promoters are fundamental to regulating gene expression but also that global epigenetic

Fig. 1. Regulation of progesterone responses in endometrium and endometriosis. For detailed explanation, see text. The lightening bolds indicate perturbations in progesterone signal transduction at different levels of control, including expression of PR isoforms (Attia et al., 2000; Wu et al., 2006a,b), chaperone proteins involved in receptor recycling and ligand binding (Hirota et al., 2008), coregulators (Aghajanova et al., 2009; Caballero et al., 2005; Suzuki et al., 2010), as well as transcriptional partners and a variety of upstream signal transduction pathways capable of modifying PR and its coregulators (Kim et al., 2007; Lee et al., 2009; Shazand et al., 2004; Wu et al., 2005; Yang et al., 2002).

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changes, which invariably occur upon commitment of stem/progenitor cells to a specific lineage, ultimately determine the subsequent cellular responses to steroid hormones.

6. Mechanisms of endometrial progesterone resistance Induction of pelvic endometriosis in animal models, such as the baboon, is sufficient to disrupt the progesterone responses in the eutopic endometrium in a manner akin to the human situation (Fazleabas, 2010). At the molecular level, it is not too difficult to envisage mechanisms that would disrupt progesterone actions in target cells in the presence of chronic pelvic inflammation. For example, inflammatory signals could induce progesterone resistance by altering the expression levels of PR isoforms, chaperone proteins like FKBP52 (Hirota et al., 2008), or coregulators such as hydrogen peroxide-inducible clone 5/androgen receptor coactivator 55 (HIC-5/ARA55; Aghajanova et al., 2009). Notably, overexpressed HIC-5/ARA55potentiates the transactivation activity of not only PR but also the androgen receptor, mineralocorticoid receptor and glucocorticoid receptor (Yang et al., 2000), indicating that downregulation of this multifaceted coactivator in the endometrium of patients with endometriosis will impact on the tissue responsiveness to multiple steroid hormones. Activation of proinflammatory transcription factors could also compete with PR for a limited pool of coregulators or disrupt the interaction between the receptor and key transcriptional partners, such as forkhead transcription factor FOXO1. Moreover, inflammation is invariable associated with free radical production and oxidative stress signals, which in turn will alter the posttranslational code of PR. In fact, there is evidence to implicate most, if not all, of these mechanisms of progesterone resistance exist in endometriosis patients, as illustrated in Fig. 1. This is not entirely surprising as progesterone signal transduction is tightly controlled at different levels, albeit by interconnected mechanisms. While the evidence in support of the paradigm outlined above seems compelling, it does not entirely explain the concept of progesterone resistance in the eutopic endometrium in patients with endometriosis for two reasons. First, the model suggests that suppression of endometriotic lesions and associated inflammation, for example upon prolonged treatment with GnRH-analogues or after surgical ablation of the lesions, would suffice to restore normal steroid hormone responses and cure the disease, which is ostensibly not the case. Second, and as mentioned before, there is compelling evidence that endometriosis is associated with perturbed gene expression in purified eutopic endometrial stromal cells that are maintained in culture, irrespectively whether stimulated or not with progesterone or other differentiation cues (Aghajanova et al., 2010, 2011; Klemmt et al., 2006; Minici et al., 2008). These observations strongly infer that progesterone resistance is likely to be as much a consequence of changes in the epigenetic chromatin landscape of endometrial cells as the result of intrinsic defects in PR or other signal transduction pathways. Inflammatory signals are established epigenetic modifiers, which raises the possibility that cyclic menstruation is important for ‘programming’ hormonal responses in the uterus, especially prior to pregnancy (Brosens et al., 2009). Moreover, animal experiments demonstrated that induction of pelvic disease has long-lasting consequences for the chromatin landscape of eutopic endometrial cells by altering DNA methylation and histone tail modifications in proximal promoter regions of progesterone-dependent genes (Guo, 2009; Kim et al., 2007; Lee et al., 2009). For example, Kim and co-workers demonstrated that induction of endometriosis in the baboon resulted in a gradual decrease in endometrial HOXA10 expression, a homeobox transcription factor involved in endometrial development and differentiation. Importantly, this

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down-regulation was only significant 6–12 months after the induction of endometriosis and corresponded to increased methylation of the proximal promoter of HOXA10 (Kim et al., 2007). In order to have a lasting effect on uterine function, inflammatory cues associated with menstruation or endometriosis, must impact on basal endometrial cells, which contain the progenitor cells for the superficial endometrium as well as the junctional zone myometrium. It is indeed striking that the junctional zone myometrium is significantly thicker on T2-weighted MRI in patients with endometriosis when compared to age-matched controls (Kunz et al., 2007). 7. Conclusions As our understanding of nuclear receptor actions in general, and PR in specific, has expanded phenomenally in recent years, so have our insights into the pathological mechanisms that underpin endometriosis. Nuclear receptors like PR are widely viewed as pioneer factors responsible for initiating the process of chromatin remodelling near the transcriptional start sites of target genes. However, this paradigm is being profoundly challenged by novel techniques that allow genome-wide mapping of binding of nuclear receptors to DNA. Contrary to expectations, a majority of nuclear binding events do not occur proximal to transcriptional start sites of target genes but at large distances from promoters. Moreover, rather than the activated receptor inducing a permissive chromatin environment that enables transcription, most activated receptors will bind at pre-existing sites that are constitutively accessible. When extrapolated to the cycling endometrium, these observations strongly suggest that acquisition of responsiveness to differentiation signals must be preceded by, or at least occur in concert with, genome-wide remodelling of the chromatin. Thus, steroid hormone responses in the endometrium are likely much more dynamic and complex than previously appreciated and are modified by the cumulative effects of cyclic menstruation and other inflammatory signals. Within this context, ‘progesterone resistance’ may primarily reflect the actions of PR on an altered chromatin landscape shaped by chronic pelvic inflammation associated with endometriosis. Conflict of Interest The authors have none to declare. References Abdel-Hafiz, H., Takimoto, G.S., Tung, L., Horwitz, K.B., 2002. The inhibitory function in human progesterone receptor N termini binds SUMO-1 protein to regulate autoinhibition and transrepression. Journal of Biological Chemistry 277, 33950– 33956. Abdel-Hafiz, H., Dudevoir, M.L., Horwitz, K.B., 2009. Mechanisms underlying the control of progesterone receptor transcriptional activity by sumoylation. Journal of Biological Chemistry 284, 9099–9108. Aghajanova, L., Velarde, M.C., Giudice, L.C., 2009. The progesterone receptor coactivator Hic-5 is involved in the pathophysiology of endometriosis. Endocrinology 150, 3863–3870. Aghajanova, L., Horcajadas, J.A., Weeks, J.L., Esteban, F.J., Nezhat, C.N., Conti, M., Giudice, L.C., 2010. The protein kinase A pathway-regulated transcriptome of endometrial stromal fibroblasts reveals compromised differentiation and persistent proliferative potential in endometriosis. Endocrinology 151, 1341– 1355. Aghajanova, L., Velarde, M.C., Giudice, L.C., 2010. Altered gene expression profiling in endometrium: evidence for progesterone resistance. Seminars in Reproductive Medicine 28, 51–58. Aghajanova, L., Tatsumi, K., Horcajadas, J.A., Zamah, A.M., Esteban, F.J., Herndon, C.N., Conti, M., Giudice, L.C., 2011. Unique transcriptome, pathways, and networks in the human endometrial fibroblast response to progesterone in endometriosis. Biology of Reproduction 84, 801–815. Attia, G.R., Zeitoun, K., Edwards, D., Johns, A., Carr, B.R., Bulun, S.E., 2000. Progesterone receptor isoform A but not B is expressed in endometriosis. Journal of Clinical Endocrinology and Metabolism 85, 2897–2902.

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